The displacement of histones from the spermatid nucleus is the first step in a series of nucleoprotein transitions eventually leading to the protamine-containing nucleus that appears to be essential for a normally fertile mammalian sperm. The long-term objective is to elucidate the mechanisms that are important for this displacement of histones. The goal of this research is to describe the precise timing of the steps in the nucleoprotein replacement process in rat spermatids. Techniques for preparation of spermatid nuclei at well-defined steps (steps 2-4, 7-8, 9- 10, 11-12, 13-15) will be developed using rats in which the cycle of the seminiferous epithelium is synchronized by manipulation of vitamin A levels. Testes from these rats will be used as starting material for cell separation by centrifugal elutriation and equilibrium density centrifugation. The analyses will be designed to investigate several hypotheses for mechanisms of histone displacement, by polyamines or more basic proteins, and proteolysis. In the accurately staged populations of nuclei, the levels of histone modification (acetylation, phosphorylation, ubiquitination), the types of transition proteins (TP1, TP2 and others) that are present after the loss of histones, the quantitative levels of histones and transition proteins, the levels of polyamines, and the fate of the displace histones will all be characterized. Polyacrylamide gel electrophoresis will be used to perform most of the analyses of histones although other techniques in protein chemistry will be applied as well. These results will be analyzed to test which of the above hypotheses are consistent with the actual time sequence of histone displacement in the spermatids. The results will also be used to establish conditions for preliminary in vitro studies on histone displacement from rat spermatid nuclei at the appropriate step of development.